Recently, it has been shown that inactivation of estrogen receptor β (ER‐β) by gene targeting results in increased cortical bone formation in adolescent female mice. To study the possible involvement of ER‐β in the regulation of the mature skeleton, we have extended the analyses to include 1‐year‐old ER‐β knockout mice (ER‐β−/−). Male ER‐β−/− mice did not express any significant bone phenotypic alterations at this developmental stage. However, the increase in cortical bone parameters seen already in the adolescent female ER‐β−/− mice was maintained in the older females. The aged female ER‐β−/− mice further exhibited a significantly higher trabecular bone mineral density (BMD) as well as increased bone volume/total volume (BV/TV) compared with wild‐type (wt) mice. This was caused by a less pronounced loss of trabecular bone during adulthood in female ER‐β−/− mice. The growth plate width was unaltered in the female ER‐β−/− mice. Judged by the expression of the osteoclast marker tartrate‐resistant acid phosphatase (TRAP) and cathepsin K (cat K; reverse‐transcription‐polymerase chain reaction [RT‐PCR]) as well as the serum levels of C‐terminal type I collagen cross‐linked peptide, bone resorption appeared unaffected. However, an increase in the messenger RNA (mRNA) expression levels of the osteoblast marker core‐binding factor α1 (Cbfa1) suggested an anabolic effect in bones of old female ER‐β−/− mice. In addition, the mRNA expression of ER‐α was augmented, indicating a role for ER‐α in the development of this phenotype. Taken together, the results show that ER‐β is involved in the regulation of trabecular bone during adulthood in female mice and suggest that ER‐β acts in a repressive manner, possibly by counteracting the stimulatory action of ER‐α on bone formation.